This invention relates to a process for producing L-lysine in enhanced yields by fermentation. More specifically, this invention relates to a process for producing L-lysine in enhanced yields which comprises culturing an L-lysine-producing microorganism in a nutrient medium supplemented by the culture liquor of an L-leucine-producing microorganism.
L-lysine is well known as one of the essential amino acids for human and animal nutrition and is in great demand as a food and animal feed supplement.
Heretofore, there has been known a variety of processes for the fermentative production of L-lysine. The typical of those processes comprise the use of L-lysine-producing mutants of coryneform glutamic acid-producing bacteria represented by Corynebacterium glutamicum.
Coryneform glutamic acid-producing bacteria are clearly defined in the art. They are generally characterized by being ellipsoidal spheres to short rods, Gram-positive, non-sporulating and non-motile, requiring biotin and accumulating large amounts of L-glutamic acid.
Numbers of coryneform glutamic acid-producing bacteria have been already reported. They are classified depending upon the opinion of taxonomists who conducted studies on the bacteria, to the species: Corynebacterium glutamicum. Brevibacterium aminogenes, Brevibacterium divaricatum, Brevibacterium flavum, Brevibacterium lactofermentum, Brevibacterium roseum, Brevibacterium saccharolyticum, Brevibacterium immariophilium, Corynebacterium acetoacidophilum, Corynebacterium herculis, Corynebacterium lilium, Corynebacterium callunae, Microbacterium ammoniaphilum and Arthrobacter species.
However, they are taxonomically very closely related with each other as described by Abe et al in J. General and Applied Microbiology, Vol. 13, 279- 301 (1967). Coryneform glutamic acid-producing bacteria are represented by Corynebacteium glutamicum.
L-lysine-producing mutants of coryneform glutamic acid-producing bacteria are generally characterized by having at least one of the two properties resulted from gene mutation.
One of the two properties is the complete or incomplete blockage of biosynthetic pathway for the production of related amino acids. This property is recognized as a requirement for related amino acids such as L-homoserine, L-threonine, L-methionine, L-leucine, L-isoleucine, etc. or as a sensitivity to L-threonine or L-methionine. The other property is the complete or incomplete deviation from the feedback regulations by related amino acids. This property is recognized as a resistance to related amino acids such as L-lysine, L-threonine, etc. or their analogs such as S-(2-aminoethyl)-L-cysteine, etc.
The productivity of L-lysine by the L-lysineproducing mutants having the above properties is improved by combining further genetical mutations other than those described above, for example, requirements for amino acids such as L-valine, L-tyrosine, etc., vitamins such as thiamin, vitamin B.sub.12, etc. and purine bases such as adenine, guanine, etc.
Further, the productivity of L-lysine is also improved by combining genetical mutations other than those described above, for example, resistances to amino acids such as L-isoleucine, their analogs such as 2-amino- 3-methylthiobutyric acid, etc. and antibiotics such as penicillin G, polymixin B, etc.
Preferred L-lysine-producing mutants include:
Micrococcus glutamicus ATCC 13286, ATCC 13287,
brevibacterium flavum ATCC 21475, ATCC 21127.
ATCC 21128, ATCC 21517, ATCC 21518, ATCC 21528
ATCC 21529, and
Corynebacterium glutamicum ATCC 21299, ATCC 21300,
ATCC 21513, ATCC 21514, ATCC 21515, ATCC 21516,
ATCC 21527, ATCC 21544, KY 10403, KY 10031.
Most of the above-mentioned specific mutants are described in U.S. Pat. Nos. 2,979,439, 3,616,218, 3,687,810, 3,707,441, 3,708,395 and U.K. Pat. No. 1,186,988.
In producing L-lysine by culturing L-lysine-producing strains such as described above, these strains are cultured aerobically in a medium containing an assimilable carbon source, a nitrogen source, inorganic materials and other nutrients.
While studying the production of L-lysine by using an L-lysine-producing strain having a requirement for L-leucine, the present inventors used a culture liquor of an L-leucine-producing mutant as a supplement to the medium in order to satisfy the requirement for L-leucine. As the result, it was unexpectedly found that the productivity of L-lysine by the L-lysine-producing strain was remarkably increased. Further, it was found that the effect is much superior to that where free L-leucine is added to the medium in an amount corresponding to that contained in the culture liquor of the L-leucine-producing mutant.
Furthermore, it was found that the effect of the addition of the culture liquor of the L-leucine-producing mutant was obtained not only when the L-lysine-producing strain having a requirement for L-leucine but also always when L-lysine-producing strain having various properties are used.
The fermentative production of L-leucine has also been well known in the art. As in the case with the L-lysine-producing mutants, such L-leucine-producing mutants generally have at least one property selected from a requirement for related amino acids including L-isoleucine, L-methionine and L-valine and a resistance to related amino acids including L-leucine and their analogs such as .alpha. -aminobutyric acid etc. resulted from the complete or incomplete blockage of biosynthetic pathway and the complete or incomplete deviation from the feedback regulations. The productivity of L-leucine is improved by combining properties other than those described above, for example, requirement for amino acids such as L-phenylalanine, etc. Further the productivity is also improved by combining resistances to amino acids such as L-lysine, L-histidine, etc. and their analogs such as S-(2-aminoethyl)-L-cysteine, 2-thiazolealanine, etc.
Preferred L-leucine-producing mutants include:
Brevibacterium flavum (FERM-P No. 1838) ATCC 21889,
brevibacterium lactofermentum (FERM-P No. 1837) ATCC 21888,
corynebacterium glutamicum ATCC 21301-21308, ATCC 21885, ATCC 21886 (FERM-P No. 1835), and Corynebacterium acetoacidophilum (FERM-P No. 1836) ATCC 21887.
Some of the above-mentioned specific strains are described in Japanese Unexamined Patent Application Published under No. 101589/74.
According to the present invention, the yields of L-lysine are greatly improved by culturing L-lysine-producing mutants in a medium supplemented by the culture liquor of L-leucine-producing mutants. This mechanism has not been clarified yet. It has already been well known to improve the productivity of L-lysine by the addition of various amino acids. As described above, the effect of the addition of the culture liquor of L-leucine-producing mutants has been found not due to L-leucine contained in the culture liquor. Although the culture liquor of L-leucine-producing mutants contain various amino acids other than L-leucine, the effect of the addition of the culture liquor has also been found not attributable to such amino acids as described in detail hereinbelow.
Further, the effect of the addition of the culture liquor of L-leucine-producing mutants is characteristic to itself and such effect is not found when the culture liquor of L-glutamic acid-producing microorganisms is used.